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This article explains how embryo grading is used to evaluate the quality and developmental potential of embryos in IVF. By using an embryo grading system, embryologists assess embryos based on their morphology and growth patterns from the early zygote stage to the blastocyst phase. In ivf embryo grading, factors such as the expansion of the blastocyst, the inner cell mass, and the trophectoderm are analyzed to determine implantation potential. For example, a 5AA embryo represents a highly developed blastocyst with strong chances for successful pregnancy. Understanding these grading criteria helps improve embryo selection and overall IVF outcomes.
The quality of an embryo is primarily determined by the condition of the gametes and the environment in which it is cultured. Recognizing the maturity stage of the oocyte is essential for scheduling the fertilization process appropriately. Several evaluation methods have been proposed to classify embryos during the pronuclear, cleavage, and blastocyst stage (1).
In human assisted reproduction laboratories, the most commonly applied approach for evaluating embryos relies on their morphological features, which is heavily influenced by the embryologist’s experience (2). The implementation of time-lapse technology (TLT) in embryo culture has been a major change in everyday practice in IVF laboratories. The Embryology Interest Group of the Association for the Study of Reproductive Biology (ASEBIR) developed a scoring system that categorizes embryos into four grades—A, B, C, and D.
The likelihood of successful outcomes increases progressively across these categories, with grade A showing the highest and grade D the lowest success rates (3). This grading system serves as a predictive tool for assessing implantation and live birth potential (2).
Evaluating embryo development involves analyzing both morphological characteristics (appearance and structure) and morphokinetic patterns (timing and sequence of developmental events). These assessments offer complementary insights: while morphology provides a static view at specific time points, morphokinetics—especially when observed through time-lapse technology (TLT)—reveals continuous developmental behavior. By combining these two approaches, embryologists can more accurately identify embryos with higher developmental competence, better implantation potential, and a reduced likelihood of chromosomal abnormalities. The following sections outline key evaluation criteria across the major stages of embryo development, from the zygote stage to the blastocyst phase.
The key criteria for evaluating zygotes through a punctual static morphological assessment include the number of polar bodies and pronuclei, the structural features of the pronuclei, the nucleolar precursor bodies, and the presence of a cytoplasmic halo. Time-lapse technology (TLT) provides embryologists with new parameters that help analyze embryo development. The pronuclear fading time (tPNf) is often used to predict embryo features like chromosome status. Some studies found that aneuploid embryos have longer tPNf or longer intervals between tPNf and the first cleavage, while others reported no clear link between tPNf and embryo ploidy (4,5.6,)
The number of blastomeres is a crucial factor for forecasting embryo development potential. This evaluation is typically performed within specific time windows: 43–45 hours on day 2 and 67–69 hours on day 3, which aligns with standard day 3 embryo grading practices (7). Embryos that complete the second and third cell cycles within these expected time frames generally show higher implantation potential. In contrast, embryos with abnormal or slowed cell division often demonstrate reduced success rates, partly due to an increased likelihood of aneuploidy (8,9).
On day 4, morphology is the least researched aspect of embryo development. The absence of predictive morphological indicators for pregnancy is the primary reason transfers typically are not performed at the morula stage. Cellular compaction is a key parameter on day 4. Embryos that complete compaction before 79.93 h of development, before reaching blastocyst stage, show higher implantation potential (3,10).
Culturing embryos to the blastocyst stage offers valuable insights and is widely used for embryo selection. Optimal evaluation of blastocysts occurs between 114–118 hours post-insemination on day 5 and 136–140 hours on day 6. Blastocyst stage is considered a dynamic state of development.
The ASEBIR grading system classifies blastocysts into four categories (A, B, C, D), based on key criteria including the degree of expansion, the quality of the trophectoderm (the outer layer of cells in a blastocyst), and the quality of the inner cell mass (ICM). Among these, trophectoderm quality is considered the most critical determinant of overall blastocyst quality, as supported by previous studies (11,12,13).
| Grade | Degree of Expansion | Trophectoderm Quality | Inner Cell Mass (ICM) Quality |
|---|---|---|---|
| A | Fully expanded blastocyst | Uniform, cohesive epithelium with elliptic cells | Many tightly packed, cohesive cells forming a dense and distinct cluster |
| B | Expanded blastocyst, but not fully | Mostly cohesive with minor irregularities; most cells appear healthy | Several cells present but the cluster is less compact compared to Grade A |
| C | Some expansion, but still limited | Irregular epithelium, fewer cells, signs of degeneration | Few loosely grouped cells with structural irregularity |
| D | Minimal or no expansion | Irregular, degenerating cells and poor epithelial structure | Very few or degenerating cells; ICM poorly defined or absent |
Among an increasing number of freeze-all cycles, the decision of whether to freeze a blastocyst represents a critical embryology decision at the culmination of an IVF cycle. The decision to freeze a blastocyst is guided by blastocyst grading schemes. Blastocyst grading systems provide morphological criteria for different grading tiers of the inner cell mass (ICM) and trophectoderm (TE). (14).
The Gardner classification (or Gardner grading system) is a commonly used method in embryology to evaluate blastocyst-stage embryos during in vitro fertilization (IVF). It assesses both the degree of blastocyst development (expansion) and the quality of the inner cell mass (ICM) and trophectoderm (TE) (15).
| Stage | Description |
|---|---|
| 1 | Early blastocyst: blastocoel just begins to form. |
| 2 | Blastocoel occupies about half of the embryo’s volume. |
| 3 | Full blastocyst: blastocoel completely fills the embryo. |
| 4 | Expanded blastocyst: blastocoel volume larger than original embryo; zona pellucida begins thinning. |
| 5 | Hatching blastocyst: trophectoderm begins to break through the zona pellucida. |
| 6 | Hatched blastocyst: fully emerged from the zona pellucida. |
| ICM Grade | Description |
|---|---|
| A | Many tightly packed cells — a well-defined, strong and healthy inner cell mass. |
| B | Several cells present but less compact and organized than Grade A — moderate quality. |
| C | Few cells that are poorly organized or show degeneration — low quality ICM. |
| TE Grade | Description |
|---|---|
| A | Many cells forming a cohesive, uniform epithelial layer. |
| B | Fewer cells that form a looser and less uniform epithelial structure. |
| C | Very few large, irregular, or degenerating cells — poor trophectoderm quality. |
| Gardner Code | Blastocyst Stage | ICM Grade | TE Grade | Description |
|---|---|---|---|---|
| 4AA | Expanded blastocyst (Stage 4) | A (excellent ICM) | A (excellent TE) | Represents a well-developed blastocyst with high implantation potential. |
| 3BB | Full blastocyst (Stage 3) | B (moderate ICM) | B (moderate TE) | Moderate-quality embryo with average implantation chances. |
| 5BC | Hatching blastocyst (Stage 5) | B (moderate ICM) | C (poor TE) | May still be viable, but associated with lower implantation potential. |
Embryo grading plays a key role in improving IVF success. By using standardized embryo grading systems, embryologists evaluate embryo development based on morphology and growth, especially the ICM, trophectoderm, and blastocyst expansion. A high-quality embryo such as a 5AA embryo indicates strong implantation potential. Understanding these grading criteria helps select the best embryos and enhances overall IVF outcomes.
